title: Molecular basis of interactions between mitochondrial proteins and hydroxyapatite in the presence of Triton X-100, as revealed by proteomic and recombinant techniques.
abstract: Hydroxyapatite chromatography is a very important step in the purification of voltage-dependent anion channels (VDACs) and several members of solute carrier family 25 (Slc25) from isolated mitochondria. In the presence of Triton X-100, VDACs and Slc25 members present a peculiar property, i.e., a lack of interaction with hydroxyapatite, resulting in their presence in the flow-through fraction of hydroxyapatite chromatography. This property has allowed selective isolation of VDACs and Slc25 members from a mixture of total mitochondrial proteins. However, the reason why only these few proteins are selectively obtained in the presence of Triton X-100 from the flow-though fraction of hydroxyapatite chromatography has not yet been adequately understood. In this study, when we examined the protein species in the flow-through fractions by proteomic analysis, VDAC isoforms, Slc25 members, and some other membrane proteins were identified. All the mitochondrial proteins had in common high hydrophobicity over their entire protein sequences. When the proteins were fused to soluble proteins, the fused proteins showed affinity for hydroxyapatite even in the presence of Triton X-100. Based on these results, we discussed the molecular basis of the interactions between proteins and hydroxyapatite in the presence of Triton X-100.
authors.affiliation: Institute for Genome Research, University of Tokushima, Kuramotocho-3, Tokushima 770-8503, Japan. tyamamo@genome.tokushima-u.ac.jp
authors.forename: Takenori
authors.initials: T
authors.lastname: Yamamoto
authors.forename: Haruna
authors.initials: H
authors.lastname: Tamaki
authors.forename: Chie
authors.initials: C
authors.lastname: Katsuda
authors.forename: Kiwami
authors.initials: K
authors.lastname: Nakatani
authors.forename: Satsuki
authors.initials: S
authors.lastname: Terauchi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Terada
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Shinohara
mesh_terms: D000818:Animals; D017886:Durapatite / Q000737:chemistry* / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000378:metabolism; D017830:Octoxynol / Q000737:chemistry*; D020033:Protein Isoforms; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D017830:Octoxynol; D017886:Durapatite
keywords: Hydroxyapatite; Mitochondria; Proteomics; Slc25; Triton X-100; VDAC